The invention relates to a process for the production of the pure L-isomer of malic acid from neutralized fumaric acid in highly concentrated aqueous solution by biotechnical conversion, at least half of the neutralized fumarate being in the form of NH.sub.4 -fumarate.
L-Malic acid is utilized in the food and pharmaceutical industries as a buffer material, a complexing agent, an acidulant, and a moisturizer. L-Malic acid, as a natural substance, is more suitable for these areas of application than the chemically accessible DL-isomer mixture of malic acid, producible, for example, by addition of water to maleic anhydride. The D-isomer of malic acid does not occur in nature; for this reason, chemically synthesized DL-malic acid is not always acceptable for food and pharmaceutical use.
Several methods are known for the biotechnical production of L-malic acid. In some of these processes (e.g., DE-AS No. 23 63 285), special bacteria convert fumaric acid into L-malic acid by the addition of water. U.S. Pat. No. 3,922,195 teaches immobilizing the bacterial cells, as well as use of the yeast Pichia farinosa. According to DAS No. 2,363,285, the process is carried out with free bacterial cells or according to DE-AS No. 24 15 310 with the enzyme fumarase, isolated from bacterial cells.
Conversion of glucose to L-malic acid with a combination of a fungus and a bacterium has been described [J. Ferment Technol. 54 (4): 197-204 (1976)]. In this method, the fungus makes fumaric acid from glucose, and this fumaric acid is then converted into L-malic acid by the bacterium.
The enzyme fumarase, effecting conversion of fumaric acid to L-malic acid, can be isolated in special processes from biological material, preferably the cell mass of microorganisms, and then can be used, in free or immobilized form to obtain L-malic acid from fumaric acid (DAS No. 2,415,310; Czechoslovakian Pat. No. 171,990).
In several processes, a large amount of a calcium compound is added to the fermentation batch, whereby the resultant L-malic acid is precipitated during the course of the fermentation itself, as calcium malate (e.g., the process of DOS No. 1,417,033).
Processes for the biotechnical manufacture of L-malic acid using fungi are also known. In these processes, the L-malic acid is essentially obtained by biochemical degradation and/or biosynthesis from the carbon source, e.g., molasses, sugar, ethanol, acetic acid, fed to the particular fungus [J. Ferment Technol. 55 (2): 196-199 (1977)]. These processes do not start directly with fumaric acid.
In the process described in U.S. Pat. No. 3,063,910 and the equivalent GB Pat. No. 884,029, the fermentation batch contains 10-15% of a sugar, and can also contain 1-10% of an organic acid, e.g., pyruvic acid or fumaric acid. However, in this fungal method, the added organic acid does not serve as a substrate for the synthesis of L-malic acid, but rather as a reaction accelerator for the biochemical transformation of the sugar substrate into L-malic acid. This is obvious especially from claim 4 of GB Pat. No. 884,029.
Fermentative conversion of n-paraffin into L-malic acid is possible by the association of two types of yeasts, wherein one type of yeast converts paraffin into fumaric acid, and the second type of yeast converts the resultant fumaric acid into L-malic acid [Agr. Biol. Chem. 34: 1833-1838 (1970)].
The biotechnical production of L-aspartic acid from hydrocarbons with ammonium fumarate as the intermediate stage is described in U.S. Pat. No. 4,013,508, in which a bacterium associated with a fungus is used. The ammonium fumarate is converted practically totally into L-aspartic acid, in which finally thermodynamic bases for formation of L-aspartic acid and against formation of L-malic acid from ammonium fumarate are mentioned.
Other data on the biotechnical conversion of ammonium fumarate to L-aspartic acid can be found in DE-PS No. 23 45 271 and BE No. 818,480.
The known methods for the biotechnical manufacture of L-malic acid have, inter alia, the following properties:
Biotechnical processes using bacteria are more difficult to handle than those using fungi. For example, separation problems can arise due to the small size of the bacteria. Furthermore, bacteria tend to form by-products, some of which are toxic. A particularly disturbing factor militating against use of L-malic acid in the pharmaceuticals area is LPS toxin (lipopolysaccharide toxin) formed by bacteria, which has a pyrogenic effect, and from which L-malic acid can be separated only by expensive process steps, e.g., ultrafiltration. In the methods described by U.S. Pat. No. 3,922,195, succinic acid occurs in small amounts as by-product, and can be separated from malic acid only with difficulty.
When using the isolated enzyme fumarase, the primary fumarase source is microbial cell mass. Before production of L-malic acid can be started, microbial cell mass must be grown and worked up, at considerable expense, to yield fumarase.
Immobilization of living cells or of the enzyme fumarase isolated from cells is an additional process step.
When fermenting in the presence of calcium ions in high concentration, the resultant L-malic acid is precipitated as calcium malate during the course of the fermentation; this leads to problems in effecting adequate agitation and aeration of the fermentation batch.
The attainable concentration of L-malic acid is generally comparatively low and lies markedly below 100 g per liter of fermentation batch except for the reactions described in U.S. Pat. No. 3,922,195 (immobilized microorganisms) and in DOS No. 1,417,033 (high calcium concentration and bacteria as source of fumarase).
Fermentation takes longer than three days. The large capacity of the bioreactors necessitated by this low yield results in high costs for apparatus; all the more so since the reactors are of an expensive construction to enable the process to be conducted free of contamination.
Although the term "fermentation" in technical literature has a relatively large spectrum of meanings, the expression "fermentation" in the context of the present invention means the fungal bioconversion of fumarate to L-malate, catalyzed by the enzyme fumarase, by incubation of a fumarase-containing microbial biomass, under bioconversion conditions. The term "culturing" means incubating the biomass from the inoculum in a nutrient medium, under conditions conducive to proliferation of the cell mass and consumption of the nutrients.
The production of the pure L-isomer of malic acid from neutralized fumaric acid by microbial fermentation by means of freely moving microorganisms in aqueous phase and of a concentration of L-malic acid at the harvest time of 100 to 170 g per liter of fermentation liquid is described in DE-OS No. 33 10 849.8. This process is marked by great productivity. The yield amounts to more than 77% of the theoretical yield. No D-malic acid is produced. The L-malic acid is obtained in a solution, whose working up comes very close to a quality suitable for food or pharmaceutical use. In this process the main amount of fumaric acid is used in the form of its sodium and/or potassium salt. DE-OS No. 33 10 849.8 corresponds to U.S. patent application Ser. No. 592,977, filed Mar. 23, 1984, the entire disclosure of which is incorporated herein by reference.
Although this process considerably exceeds the state of the art, the object is to improve it still more.